CN106749552B

CN106749552B - WU polyoma virus recombinant antigen and preparation method and application thereof

Info

Publication number: CN106749552B
Application number: CN201611023519.7A
Authority: CN
Inventors: 陆学东; 张银辉; 何英
Original assignee: Eighth Affiliated Hospital of Sun Yat-sen University
Current assignee: Eighth Affiliated Hospital of Sun Yat-sen University
Priority date: 2016-11-21
Filing date: 2016-11-21
Publication date: 2020-04-07
Anticipated expiration: 2036-11-21
Also published as: CN106749552A

Abstract

The invention provides a WU polyoma virus recombinant antigen and a preparation method of the WU polyoma virus recombinant antigen, the method comprising the following steps: linking the WU polyoma virus VP2 nucleic acid fragment and the WU polyoma virus STAg nucleic acid fragment to obtain VP2 ‑STAg nucleic acid fragment is loaded into PGEX‑20T plasmid to obtain PGEX‑20T‑VP2‑STAg recombinant plasmid and then transferred to Escherichia coli, and induced to express protein. After protein purification, WU polyoma virus recombinant antigen is obtained. The WU polyoma virus recombinant antigen prepared by the preparation method of the invention has good antigenicity and can be used for the detection of antibodies in children infected with WUPyV, and also can be used for epidemiological investigation and research of WUPyV infection in the population.

Description

WU polyoma virus recombinant antigen and preparation method and application thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to a WU polyoma virus recombinant antigen and a preparation method and application thereof.

Background

WU polyomavirus (WU polyomavirus, WUPyV) is a new polyomavirus found by us and australian scientists in 2007 in respiratory specimens of 1 infant with pneumonia at 3 years old. The research finds that WUPyV is widely distributed around the world, the existence of the WUPyV is detected in various tissues, body fluids and secretions of a human body, and a large number of molecular biology and limited serological research results show that the WUPyV mainly infects infants and young children through respiratory tracts and causes serious lower respiratory tract diseases such as severe pneumonia and the like in partial cases.

1. Molecular biological characteristics of WUPyV:

the WUPyV whole gene has the length of 5229bp (or 5228bp), is a closed circular double-stranded DNA molecule and has the GC content of 39%. The genome comprises an early coding region, encoding the large T antigen (LTAg), the small T antigen (STAg), and a late coding region, encoding capsid proteins VP1, VP2, and VP3, in the other chain, separated by non-coding regulatory regions (see figure 1).

Both DNA synthesis and transcription start in the regulatory region and proceed in opposite directions, controlling early and late gene transcription and DNA replication, respectively.

The gene variation of WUPyV is considered to be mainly in the VP1 region by the research of Venter et al, the VP2 and STAg genes are less varied, and the end of the regulatory region-VP 2 beginning (287-722nt) is the most conserved region of WUPyV. WUPYV is firstly detected in south China by the subject group, and the genome identification result shows that the Guangdong strain (GenBank sequence number GQ 926975-926980) has high homology with the WUPYV strain detected and identified in Gaynor, Korea, Zhejiang and Beijing areas of China.

2. The current research situation is as follows:

to date, no complete WUPyV particles have been isolated, no in vitro or in vivo viral propagation has been reported, and no corresponding cell lines or animal models are available, and it is to be verified whether the gene-encoding open reading frame predicted from the genome can be actually expressed during the course of viral infection.

Most WUPyV is detected from respiratory secretions of infants and children of low age, and the detection rate of the respiratory tract of adults is very low. The WUPyV detection rate in the body of the person with low immune function is reported differently. Neske et al found that the WUPyV IgG antibody was present in about 89% of healthy blood donors.

WUPyV assays currently rely entirely on molecular biology methods.

WUPYV serology research is few in reports, VP1 capsid protein is mainly expressed at present, and research on expression of VP2, VP3 and STAg protein is not reported yet. That is, human awareness of WUPyV is extremely limited, and many studies are limited to epidemiological studies. WUPYV is known to mainly infect infants and people with low immune function, but the transmission path, latent tissues or organs, latent period, cell entering mode, immune reaction mode after human body is infected with virus, pathogenicity of virus, whether human body tumor is caused like other polyoma virus and other problems are yet to be determined, and especially pathogenicity needs to be determined firstly.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: provides a WU polyoma virus recombinant antigen with high sensitivity and specificity, a preparation method and application thereof.

In order to solve the technical problems, the invention adopts the technical scheme that: a WU polyoma virus recombinant antigen comprises amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO. 2.

The WU polyoma virus recombinant antigen is obtained by expressing a WU polyoma virus VP2 nucleic acid fragment with a nucleotide sequence shown as SEQ ID NO.3 and a WU polyoma virus STAg nucleic acid fragment with a nucleotide sequence shown as SEQ ID NO. 4.

The invention also provides a preparation method of the WU polyoma virus recombinant antigen, which comprises the following steps:

step 1, linking a WU polyoma virus VP2 nucleic acid fragment with a nucleotide sequence shown as SEQ ID NO.3 and a WU polyoma virus STAg nucleic acid fragment with a nucleotide sequence shown as SEQ ID NO.4 to obtain a VP2-STAg nucleic acid fragment;

step 2, loading the VP2-STAg nucleic acid fragment into a PGEX-20T plasmid to obtain a PGEX-20T-VP2-STAg recombinant plasmid;

and 3, transferring the PGEX-20T-VP2-STAg recombinant plasmid into escherichia coli, inducing the PGEX-20T-VP2-STAg recombinant plasmid to express protein, and purifying the protein to obtain the WU polyoma virus recombinant antigen.

The invention also provides application of the WU polyoma virus recombinant antigen in preparation of vaccines or medicines for preventing or treating WU polyoma virus infection.

The invention has the beneficial effects that: the WU polyoma virus VP2 nucleic acid fragment and the chimeric fragment formed by linking the WU polyoma virus STAg nucleic acid fragment construct an expression vector, and the expressed recombinant protein has good sensitivity and high specificity.

Drawings

Fig. 1 is a diagram of the genomic structural pattern of WU polyoma virus (Gaynor, 2007).

FIG. 2 is a diagram showing PCR product profiles obtained by using primers for the nucleic acid fragment of the WU polyoma virus VP2 according to the embodiment of the present invention.

FIG. 3 is a PCR product map obtained by using primers of the nucleic acid fragment of WU polyoma virus STAg in the embodiment of the invention.

FIG. 4 is an identification map of PGEX-20T-VP2-STAg recombinant plasmid in the example of the present invention.

FIG. 5 shows the SDS-PAGE and immunoblotting results of protein antigen purified by GST-Resin purification column according to the present invention.

Detailed Description

In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.

The most key concept of the invention is as follows: the expression vector is constructed by the chimeric fragment formed by linking the WU polyoma virus VP2 nucleic acid fragment and the WU polyoma virus STAg nucleic acid fragment, and the expressed recombinant protein has good sensitivity and high specificity.

The invention provides a WU polyoma virus recombinant antigen which comprises amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO. 2.

The invention also provides a recombinant vector which comprises nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO. 4.

In the step 1, the WU polyoma virus VP2 nucleic acid fragment with the nucleotide sequence shown as SEQ ID NO.3 is prepared from the primer group with the base sequence shown as SEQ ID NO.5 and SEQ ID NO.6, and the WU polyoma virus STAg nucleic acid fragment with the nucleotide sequence shown as SEQ ID NO.4 is prepared from the primer group with the base sequence shown as SEQ ID NO.7 and SEQ ID NO. 8.

The method specifically comprises the following steps:

step 1, carrying out PCR reaction on a primer group with a base sequence shown as SEQ ID NO.5 and SEQ ID NO.6 to obtain a nucleic acid fragment of the WU polyoma virus VP 2; carrying out PCR reaction on a primer group with a base sequence shown as SEQ ID NO.5 and SEQ ID NO.6 to obtain a nucleic acid fragment of WU polyoma virus (STAG);

step 2, carrying out blunt end linkage on the WU polyoma virus VP2 nucleic acid fragment and the WU polyoma virus STAg nucleic acid fragment obtained in the step 1 by using T4DNA ligase, and amplifying by using a primer group with an alkali base sequence such as SEQ ID NO.5 and SEQ ID NO.8 to obtain a VP2-STAg nucleic acid fragment;

step 3, carrying out double enzyme digestion on the VP2-STAg nucleic acid fragment and the PGEX-20T plasmid by using BamH I and Xbal respectively, then carrying out ligation reaction by using T4DNA ligase, and obtaining PGEX-20T-VP2-STAg recombinant plasmid after transformation and screening of a ligation product;

and 4, transferring the PGEX-20T-VP2-STAg recombinant plasmid into a competent cell of E and coli strain BL21, expressing recombinant protein after IPTG induction, and purifying the protein to obtain the WU polyoma virus recombinant antigen.

Examples

Design, expression and identification of WUPyV VP2-STAg chimeric protein

The VP2 nucleic acid fragment was designed based on the WUPyV gene sequence (GQ926979.1) cloned from GD-WU816 in this laboratory sample, in order to avoid the nucleic acid overlap of VP3 and increase the specificity of VP2 antigen; likewise, the STAg nucleic acid fragment was designed to avoid the nucleic acid overlap of LTAg, increasing the specificity of the LTAg antigen. And linking the VP2 nucleic acid fragment and the STAg nucleic acid fragment together to obtain the nucleic acid fragment sequence of the VP2-STAg chimeric protein antigen.

1. VP2 nucleic acid fragment primers: (GQ926979.0 sequence, 574-1002nt)429bp

F (SEQ ID NO.5, containing the BamH I cleavage site):

5’-CGCGGATCCCGCATGGGCATATTGCTTGCTGTGCCTGAA-3’；

R(SEQ ID NO.6)：

5’-TCCTGGGTAGGGGGCGTGGAGG-3’

nucleotide sequence of VP2 nucleic acid fragment (SEQ ID NO. 3):

atgggca tattgcttgc tgtgcctgaa ataattgctg catctgtagc tggaggagcagaggcactat caattgctgg atctggagct gcaatagcaa ctggtgaagg tttagctgct cttggtgggcttacagagtc agcagcacta ttaggggaaa ctgttgaaat atctgaagca gctgctactg tactaacaaaagtacctgag cttgtaactg taacacaagg tgtaacagca gctgtacaag ggggtgcagg tcttgtaggtggtatatata cagctttagc agcagatcgc cctggggacc tgcctgcgag taccccaaca ggaagtccaagtggactaca tccccccgca ggatacaatc cccaaggagg tggacttaat atccagtcca tccacaagcccctccacgcc ccctacccag ga

VP2 nucleic acid fragment (SEQ ID NO.1, 143 AA):

MGILLAVPEI IAASVAGGAE ALSIAGSGAA IATGEGLAAL GGLTESAALL GETVEISEAAATVLTKVPEL VTVTQGVTAA VQGGAGLVGG IYTALAADRP GDLPASTPTG SPSGLHPPAG YNPQGGGLNIQSIHKPLHAP YPG

molecular weight: 13564.41Daltons

PCR reaction system and product obtaining: the total volume of the PCR reaction was 25. mu.l, where 1. mu.l of template, 3pmol each of the upstream and downstream primers, 0.5. mu.l of 10mmol/L dNTP mix, 10 × High Fidelity PCR buffer with MgCl22.5. mu.l, 0.15. mu.l of High Fidelity PCR enzyme mix, made up to 25. mu.l with sterile water. PCR parameters are all pre-denaturation at 94 ℃ for 3 min; denaturation at 94 deg.C for 1min, annealing at 55 deg.C for 1min, and extension at 72 deg.C for 90sec, and circulating for 30 times; finally, extension is carried out for 10min at 72 ℃. The PCR product was electrophoresed on 1% agarose gel, the fragment of the target product was rapidly excised under UV light, the gel was cut up, DNA was recovered using AxyPrep DNA gel recovery kit, the procedure was as described above, eluted with 30. mu.l of sterile deionized water, and stored at-20 ℃ for future use. The PCR product map is shown in FIG. 2. The PCR product was determined to be correct by commercial sequencing.

2. STAg nucleic acid fragment primers: (GQ926979.0 sequence, 4573-5157nt)585bp

F(SEQ ID NO.7)：

5’-ATGGATAAAACTTTGTCCAGAAATGAA-3’

R (SEQ ID NO.8, containing Xbal cleavage site):

5’-CTAGGTCTAGACTATTACCTGGTTAAGCCAACCCC-3’

nucleotide sequence of STAg nucleic acid fragment (SEQ ID NO.4, 585 bp):

atggataaaactttgtccagaaatgaagcaaaagaacttatgcagctgctgggtcttgatatgacctgctggggaaatttaccactaatgagaacaaaataccttagcaaatgcaaagaatttcatcctgacaaagggggaaatgaggaaaaaatgaaaaagcttaattctttatatttaaaactgcaagagtgtgttagtacagtgcaccaactaaatgaagaagaagatgaagtgtggagctcttcacaggtagaatgcacagaattgtgctgtaactttccccctagaaagtacaggcttgttggagaagtttatggtgatgtttttgaagagtatattttaaaagactgggacatatgcttaaaggggttttattatttgtgtaattgtttttactgctttttagacaagcgccacaagcaaaaatataaaatatttagaaaacctccaatgtggatagagtgttactgctacaggtgctatagagagtggtttggctttgaaattagtgcagaaacatttttttactggaaaaagattatatttcttacaaccatgcaaggggttggcttaaccaggtaa

amino acid sequence obtained from STAg nucleic acid fragment (SEQ ID NO.2, 195 AA):

MDKTLSRNEAKELMQLLGLDMTCWGNLPLMRTKYLSKCKEFHPDKGGNEEKMKKLNSLYLKLQECVSTVHQLNEEEDEVWSSSQVECTELCCNFPPRKYRLVGEVYGDVFEEYILKDWDICLKGFYYLCNCFYCFLDKRHKQKYKIFRKPPMWIECYCYRCYREWFGFEISAETFFYWKKIIFLTTMQGVGLTR

molecular weight: 23466.41Daltons

PCR reaction system and product obtaining: the total volume of the PCR reaction was 25. mu.l, where 1. mu.l of template, 3pmol each of the upstream and downstream primers, 0.5. mu.l of 10mmol/L dNTP mix, 10 × High Fidelity PCR buffer with MgCl22.5. mu.l, 0.15. mu.l of High Fidelity PCR enzyme mix, made up to 25. mu.l with sterile water. PCR parameters are all pre-denaturation at 94 ℃ for 3 min; denaturation at 94 deg.C for 1min, annealing at 55 deg.C for 1min, and extension at 72 deg.C for 90sec, and circulating for 30 times; finally, extension is carried out for 10min at 72 ℃. The PCR product was electrophoresed on 1% agarose gel, the fragment of the target product was rapidly excised under UV light, the gel was cut up, DNA was recovered using AxyPrep DNA gel recovery kit, the procedure was as described above, eluted with 30. mu.l of sterile deionized water, and stored at-20 ℃ for future use. The PCR product map is shown in FIG. 3. The PCR product was determined to be correct by commercial sequencing.

3. Construction of the VP2 nucleic acid fragment and STAg nucleic acid fragment linkage and expression vector:

t4DNA ligase was used for conventional blunt end ligation, according to the kit instructions. After ligation, the VP2-STAg nucleic acid fragment was amplified using VP2 upstream primer (containing BamH I cleavage site) and STAg downstream primer (containing Xbal cleavage site) to give a 1101bp chimeric fragment. After double digestion, the plasmid was filled into PGEX-20T plasmid by a conventional method.

Obtaining of recombinant plasmid

(1) PCR product and PGEX-20T plasmid digestion

The target DNA VP2-STAg nucleic acid fragment and plasmid DNA were subjected to double digestion with BamH I and Xbal, respectively, in a 20. mu.l reaction system: 10 XBuffer MC (MVLTI-CORETM Buffer) 2. mu.l, esterified BSA (10. mu.g/. mu.l), PCRDNA/plasmid DNA 5. mu.l, BamHI 0.3. mu.l, Xbal 0.3. mu.l, sterile deionized water 12.2. mu.l was added, 37 ℃, 4.5 h. And (3) after the reaction is finished, performing 1% agarose gel electrophoresis, observing the enzyme digestion result under an ultraviolet lamp, cutting a double-enzyme digestion strip, and recovering DNA by using an AxyPrepDNA gel recovery kit, wherein the method is the same as the method.

(2) PCR product and plasmid DNA ligation

Ligation reaction was performed using T4DNA ligase, VP2-STAg DNA and plasmid DNA as follows 10: 1-3: 1, and 10. mu.l of a reaction system: plasmid DNA 2.5. mu.l, VP2-STAg DNA 6.0. mu.l, 10 XT 4DNA Ligase buffer 1.0. mu.l, T4DNA Ligase 0.5. mu.l, ligated overnight at 16 ℃. The ligation was stored at-20 ℃ until use.

(3) Transformation of

Add 5. mu.l of ligation product to ice-precooled TOP10 competent cells, mix well, ice for 30min, add plasmid DNA to competent cells as positive control, and competent cells as negative control. The cells were subjected to heat shock at 42 ℃ for 90sec, taken out, rapidly cooled in ice for 2min, added with 300. mu.l of LB medium, and cultured at 37 ℃ for 1h with shaking. The transformation mixture was plated on LB plates containing 100. mu.g/ml ampicillin and incubated overnight at 37 ℃.

(4) Screening and detection of recombinant plasmid

The monoclonal colony on the LB-ampicillin screening plate is picked up and inoculated in 3ml LB culture solution containing 100 mug/ml ampicillin, shaking culture is carried out overnight at 37 ℃, plasmid miniprep kit of Tiangen is used for operation, plasmid is extracted, 60 mul sterilized deionized water is used for elution, enzyme digestion detection is carried out by BamH I and Xbal endonuclease, and recombinant plasmid is screened. The enzyme digestion identification of the constructed PGEX-20T-VP2-STAg expression plasmid is shown in FIG. 4.

4. PGEX-20T-VP2-STAg antigen expression and purification:

the PGEX-20T-VP2-STAg recombinant plasmid is transferred into a competent cell of E, coli strain BL21(DE3) by a conventional method, GST-VP2-STAg recombinant antigen is expressed after IPTG induction, and the molecular weight is about 63.5KD (GST is about 26.44 KD).

(1) Inducible expression of recombinant proteins

Mu.l of screening positive recombinant plasmid was added to precooled 25. mu.l of competent cells of expression strain BL21(DE3), mixed well and ice-cooled for 30min, then heat shocked at 42 ℃ for 90sec, rapidly kept on ice for 2min, added to 300. mu.l of LB medium, shaken at 37 ℃ for 1h, the transformation mixture was spread on LB medium plate containing 100. mu.g/ml ampicillin and cultured overnight at 37 ℃. Selecting 3-4 clone colonies in 5ml LB culture solution containing 100. mu.g/ml ampicillin, shaking for 3h at 37 deg.C, determining OD600 at about 0.6, taking out 2ml bacterial solution as control of induced bacteria, and shaking at 37 deg.C; taking out 2ml of bacterial liquid, adding 2 mu L of 1mol/L IPTG (IPTG final concentration is 1mmol/L) into a sterilization test tube, shaking for 3h at 37 ℃, centrifugally collecting the bacterial liquid, discarding the liquid, adding 50 mu L of sterile deionized water into the precipitate, uniformly mixing, adding 50 mu L of 2 multiplied Loading Buffer, uniformly mixing, boiling in boiling water for 5min, and centrifuging for 10min at 12000 g; mu.l of the supernatant was subjected to SDS-PAGE (10% separation gel, 6% concentration gel). Staining with Coomassie Brilliant blue R-250 for 30 min; decolorizing the decolorizing solution until the background is clear, and scanning and imaging by using a computer gel scanner system.

(2) Western-Blot detection of expression products

Performing SDS-PAGE on the whole induced bacteria; according to the product instruction of Bio-Rad company, a gel is close to the cathode side of an electrode, a polyvinylidene fluoride (PVDF) membrane is close to the anode side, a transfer buffer solution is 480mmol/L Tris-390mmol/L-1 mmol/LSDS-glycine-20% methanol, 100mA is used for electrotransfer for 1h, protein is transferred from the gel to the PVDF membrane, after the electrotransfer is finished, the PVDF membrane is taken out and is sealed by a 3% skimmed milk shaking table at room temperature for 2h to seal non-specific binding sites, a mouse anti-GST monoclonal antibody (5 mu L/ml) is added, the reaction is carried out for 1h at the shaking table room temperature, the membrane is washed by a washing solution at the room temperature for 4 times, each time is 5min, goat anti-mouse IgG (produced by Sigma company) diluted by 3% skimmed milk is added (diluted according to 1: 5000) and is reacted for 1h at the shaking table at the room temperature, and the reagent 2 and carbamide peroxide (100, shaking at room temperature for 5min, and washing with distilled water to terminate color reaction. SDS-PAGE and immunoblotting of protein antigens purified by GST-Resin column are shown in FIG. 5.

5. Preliminary application of VP2-STAg recombinant antigen:

the purified WUPYV VP2-STAg recombinant antigen was coated on the plate, and serum samples of patients positive for WUPYV infection (positive for PCR nucleic acid detection), healthy children control serum samples and adult control serum samples were tested by a routine ELISA test. The results of the alignment with VP2 and STAg antigens are shown in Table 1: IgG antibody detection comparison results of WUPYV VP2-STAg, VP2 and STAg recombinant proteins.

TABLE 1

The data show that the VP2-STAg recombinant protein has good antigenicity, can be used for detecting IgG antibodies of WUPYV infected children, and has a detection rate of 34.62%, the detection rate of IgG antibodies of a child control group is 52.50%, and the detection rate of the WUPYVIgG antibodies can reach more than 70% for adults with the age. It is suggested that WUPyV antibodies may have protective functions and are susceptible to disease only when infants are first infected with WUPyV. The sensitivity and specificity of the VP2-STAg recombinant protein are higher than those of the VP2 and STAg recombinant protein which are used independently,

in conclusion, the WU polyoma virus recombinant antigen prepared by the preparation method has good antigenicity, can be used for detecting the IgG antibody of a WU PyV infected child, and has the detection rate of 34.62 percent, the detection rate of the child IgG antibody of 52.50 percent and the detection rate of the WU PyV IgG antibody of more than 70 percent for adults along with the age.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.

SEQUENCE LISTING

<110> Shenzhen Shentian region people hospital

<120> WU polyoma virus recombinant antigen and preparation method and application thereof

<130>2016

<160>8

<170>PatentIn version 3.5

<210>1

<211>143

<212>PRT

<213> Artificial sequence

<400>1

Met Gly Ile Leu Leu Ala Val Pro Glu Ile Ile Ala Ala Ser Val Ala

1 5 10 15

Gly Gly Ala Glu Ala Leu Ser Ile Ala Gly Ser Gly Ala Ala Ile Ala

20 25 30

Thr Gly Glu Gly Leu Ala Ala Leu Gly Gly Leu Thr GluSer Ala Ala

35 40 45

Leu Leu Gly Glu Thr Val Glu Ile Ser Glu Ala Ala Ala Thr Val Leu

50 55 60

Thr Lys Val Pro Glu Leu Val Thr Val Thr Gln Gly Val Thr Ala Ala

65 70 75 80

Val Gln Gly Gly Ala Gly Leu Val Gly Gly Ile Tyr Thr Ala Leu Ala

85 90 95

Ala Asp Arg Pro Gly Asp Leu Pro Ala Ser Thr Pro Thr Gly Ser Pro

100 105 110

Ser Gly Leu His Pro Pro Ala Gly Tyr Asn Pro Gln Gly Gly Gly Leu

115 120 125

Asn Ile Gln Ser Ile His Lys Pro Leu His Ala Pro Tyr Pro Gly

130 135 140

<210>2

<211>194

<212>PRT

<213> Artificial sequence

<400>2

Met Asp Lys Thr Leu Ser Arg Asn Glu Ala Lys Glu Leu Met Gln Leu

1 5 10 15

Leu Gly Leu Asp Met Thr Cys Trp Gly Asn Leu Pro Leu Met Arg Thr

20 25 30

Lys Tyr Leu Ser Lys Cys Lys Glu Phe His Pro Asp Lys Gly Gly Asn

35 40 45

Glu Glu Lys Met Lys Lys Leu Asn Ser Leu Tyr Leu Lys Leu Gln Glu

50 55 60

Cys Val Ser Thr Val His Gln Leu Asn Glu Glu Glu Asp Glu Val Trp

65 70 75 80

Ser Ser Ser Gln Val Glu Cys Thr Glu Leu Cys Cys Asn Phe Pro Pro

85 90 95

Arg Lys Tyr Arg Leu Val Gly Glu Val Tyr Gly Asp Val Phe Glu Glu

100 105 110

Tyr Ile Leu Lys Asp Trp Asp Ile Cys Leu Lys Gly Phe Tyr Tyr Leu

115 120 125

Cys Asn Cys Phe Tyr Cys Phe Leu Asp Lys Arg His Lys Gln Lys Tyr

130 135 140

Lys Ile Phe Arg Lys Pro Pro Met Trp Ile Glu Cys Tyr Cys Tyr Arg

145 150 155 160

Cys Tyr Arg Glu Trp Phe Gly Phe Glu Ile Ser Ala Glu Thr Phe Phe

165 170 175

Tyr Trp Lys Lys Ile Ile Phe Leu Thr Thr Met Gln Gly Val Gly Leu

180 185 190

Thr Arg

<210>3

<211>429

<212>DNA

<213> Artificial sequence

<400>3

atgggcatat tgcttgctgt gcctgaaata attgctgcat ctgtagctgg aggagcagag 60

gcactatcaa ttgctggatc tggagctgca atagcaactg gtgaaggttt agctgctctt 120

ggtgggctta cagagtcagc agcactatta ggggaaactg ttgaaatatc tgaagcagct 180

gctactgtac taacaaaagt acctgagctt gtaactgtaa cacaaggtgt aacagcagct 240

gtacaagggg gtgcaggtct tgtaggtggt atatatacag ctttagcagc agatcgccct 300

ggggacctgc ctgcgagtac cccaacagga agtccaagtg gactacatcc ccccgcagga 360

tacaatcccc aaggaggtgg acttaatatc cagtccatcc acaagcccct ccacgccccc 420

tacccagga 429

<210>4

<211>585

<212>DNA

<213> Artificial sequence

<400>4

atggataaaa ctttgtccag aaatgaagca aaagaactta tgcagctgct gggtcttgat 60

atgacctgct ggggaaattt accactaatg agaacaaaat accttagcaa atgcaaagaa 120

tttcatcctg acaaaggggg aaatgaggaa aaaatgaaaa agcttaattc tttatattta 180

aaactgcaag agtgtgttag tacagtgcac caactaaatg aagaagaaga tgaagtgtgg 240

agctcttcac aggtagaatg cacagaattg tgctgtaact ttccccctag aaagtacagg 300

cttgttggag aagtttatgg tgatgttttt gaagagtata ttttaaaaga ctgggacata 360

tgcttaaagg ggttttatta tttgtgtaat tgtttttact gctttttaga caagcgccac 420

aagcaaaaat ataaaatatt tagaaaacct ccaatgtgga tagagtgtta ctgctacagg 480

tgctatagag agtggtttgg ctttgaaatt agtgcagaaa cattttttta ctggaaaaag 540

attatatttc ttacaaccat gcaaggggtt ggcttaacca ggtaa 585

<210>5

<211>39

<212>DNA

<213> Artificial sequence

<400>5

cgcggatccc gcatgggcat attgcttgct gtgcctgaa 39

<210>6

<211>22

<212>DNA

<213> Artificial sequence

<400>6

tcctgggtag ggggcgtgga gg 22

<210>7

<211>27

<212>DNA

<213> Artificial sequence

<400>7

atggataaaa ctttgtccag aaatgaa 27

<210>8

<211>35

<212>DNA

<213> Artificial sequence

<400>8

ctaggtctag actattacct ggttaagcca acccc 35

Claims

1. A WU polyoma virus recombinant antigen characterized by: is obtained by connecting the amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2 in sequence.

2. The WU polyomavirus recombinant antigen of claim 1, which is characterized by: is obtained by expressing a nucleic acid segment with a nucleotide sequence shown as SEQ ID NO.3 and a nucleic acid segment with a nucleotide sequence shown as SEQ ID NO. 4.

3. A preparation method of a WU polyoma virus recombinant antigen is characterized by comprising the following steps:

4. The method for preparing a WU polyoma virus recombinant antigen as defined in claim 3, wherein in step 1, the nucleic acid fragment of VP2 having a nucleotide sequence as shown in SEQ ID No.3 is prepared from the primer set having a base sequence as shown in SEQ ID No.5 and SEQ ID No.6, and the nucleic acid fragment of STAG having a nucleotide sequence as shown in SEQ ID No.4 is prepared from the primer set having a base sequence as shown in SEQ ID No.7 and SEQ ID No. 8.

5. The method of producing a WU polyoma virus recombinant antigen as claimed in claim 3, comprising the steps of:

step 1, carrying out PCR reaction on a primer group with a base sequence shown as SEQ ID NO.5 and SEQ ID NO.6 to obtain a nucleic acid fragment of the WU polyoma virus VP 2; carrying out PCR reaction on a primer group with a base sequence shown as SEQ ID NO.7 and SEQ ID NO.8 to obtain a nucleic acid fragment of WU polyoma virus (STAG);

6. Use of the WU polyomavirus recombinant antigen of any one of claims 1 to 5, wherein: is used for preparing vaccines or medicines for preventing or treating WU polyoma virus infection.